Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry

Abstract

Mass spectrometry plays a central role in the characterisation of modified nucleotides, but pseudouridine is a mass-silent post-transcriptional modification and hence not detectable by direct mass spectrometric analysis. We show by the use of matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry that pseudouridines in tRNA can be specifically cyanoethylated by acrylonitrile without affecting the uridines. The tRNA was cyanoethylated and then subjected to digestion with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by mass spectrometric comparison of untreated and acrylonitrile-treated samples, where the addition of one acrylonitrile resulted in a mass increment of 53.0 Da. The exact modified nucleotide could be identified by tandem mass spectrometry on the cyanoethylated digestion fragment. The methodology was used to identify additional one 4-thiouridine and one pseudouridine in tRNA(TyrII) from Escherichia coli. Furthermore, we observed that RNase A is highly tolerant towards nucleotide modifications, only being inhibited by 2'-O-methylation, whereas RNase T1 cleavage is affected by most nucleotide modifications.

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

“…To obtain more information on the selectivity of CMC derivatization of specific posttranscriptional modifications such as 2-methylthio-N 6 -isopentenyladenosine and queuosine, tRNA TyrII was digested with RNase A. RNase A selectively cleaves RNAs after pyrimidine residues and has been found to cleave after 5-methylcytidine, 5-methyluridine, dihydrouridine, pseudouridine and 4-thiouridine in tRNA [12]. Thus, for the tRNA TyrII sample, the 2-methylthio-N 6 -isopentenyladenosine (ms 2 i 6 A) at position 38 should be detected in the RNase A digestion product (37)-A[ms 2 i 6 A]AΨ-(39) while the queuosine (Q) residue at position 35 will not be detected because it would be present in a dimer (5′-QU-3′) whose m/z value is too low to accurately characterize with these MALDI conditions.…”

“…In related work, alternative chemical derivatizations of pseudouridine have been used prior to mass spectrometric detection [12,13]. As with the CMC-based approach, these derivatization strategies place a mass tag on pseudouridine that facilitates its detection by mass spectrometry.…”

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

“…Acrylonitrile-treated RNA-To confirm the CMCT derivatization results, an alternative derivatization strategy was explored based on the specificity of acrylonitrile for pseudouridine (30,31). LC-MS analysis of the acrylonitrile-treated T1 digest revealed the presence of triply charged oligonucleotide anions at m/z 1356.…”

“…Recently, CMCT-based derivatization has been adapted to high throughput next generation sequencing to detect dynamic pseudouridylations in mRNAs (24,25). Mass spectrometrybased RNA modification mapping in combination with chem-ical derivatization is another site-specific strategy (26) wherein a base-specific ribonuclease generates a mixture of oligoribonucleotides whose pseudouridines are chemically derivatized with either CMCT (27)(28)(29) or acrylonitrile (30,31) for detection.…”

“…Prior reports discuss the possibility of an additional pseudouridine (31) or off-target derivatization (28), but until now, those possibilities remained uninvestigated. Here we have identified the additional pseudouridine derivatization site as residing at position 35 in the anticodon.…”